Meishen Ren, Hong Mei, Ming Zhou, Zhen F. Fu, Heyou Han, Dingren Bi, Fuhu Peng and Ling Zhao. Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques[J]. Virologica Sinica, 2021, 36(2): 220-230. doi: 10.1007/s12250-020-00323-1
Citation: Meishen Ren, Hong Mei, Ming Zhou, Zhen F. Fu, Heyou Han, Dingren Bi, Fuhu Peng, Ling Zhao. Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques .VIROLOGICA SINICA, 2021, 36(2) : 220-230.  http://dx.doi.org/10.1007/s12250-020-00323-1

通过CRISPR技术开发高灵敏度的非洲猪瘟检测方法

cstr: 32224.14.s12250-020-00323-1
  • 通讯作者: 赵凌, lingzhao@mail.hzau.edu.cn, ORCID: http://orcid.org/0000-0003-0569-8105
  • 收稿日期: 2020-05-12
    录用日期: 2020-10-20
    出版日期: 2021-01-07
  • 非洲猪瘟(ASF)是一种由非洲猪瘟病毒(ASFV)引起的传染病,其临床症状为高烧,大出血和高死亡率,对全球猪业和粮食安全构成威胁。对ASFV的隔离和控制对于防止养猪业受到ASFV损害至关重要。这项研究中开发了一种基于重组酶聚合酶扩增(RPA)-CRISPR的核酸检测方法来诊断ASF。作为一种高度灵敏的方法,RPA-CRISPR可以通过定量实时PCR(qPCR)检测通过CRISPR-lwCas13a(也称为C2c2)的并行切割引起的荧光信号,该方法甚至可以检测到低至单个拷贝的ASFV质粒和基因组DNA,并且具有比传统qPCR方法相同甚至更高的灵敏度。本研究也开发了一种试纸条与RPA-CRISPR结合用于ASFV检测,具有与TaqMan qPCR相同的灵敏度。同样,RPA-CRISPR能够将ASFV基因组DNA与其他猪病病毒的DNA / RNA区别开,而没有任何交叉反应性。此诊断方法也可用于诊断ASFV临床DNA样品,对于ASFV阳性和阴性样品的符合率均为100%。作为一种高度灵敏的检测方法,RPA-CRISPR在养猪业和食品安全领域均具有对ASFV进行临床检疫的巨大潜力。

Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

  • Corresponding author: Ling Zhao, lingzhao@mail.hzau.edu.cn
  • ORCID: http://orcid.org/0000-0003-0569-8105
  • Received Date: 12 May 2020
    Accepted Date: 20 October 2020
    Published Date: 07 January 2021
  • African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) with clinical symptoms of high fever, hemorrhages and high mortality rate, posing a threat to the global swine industry and food security. Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection. In this study, a recombinase polymerase amplification (RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF. As a highly sensitive method, RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lwCas13a (previously known as C2c2) through quantitative real-time PCR (qPCR) and has the same or even higher sensitivity than the traditional qPCR method. A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of TaqMan qPCR. Likewise, RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity. This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100% for both ASFV positive and negative samples. RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.


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    Development of A Super-Sensitive Diagnostic Method for African Swine Fever Using CRISPR Techniques

      Corresponding author: Ling Zhao, lingzhao@mail.hzau.edu.cn
    • 1. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China
    • 2. Key Laboratory of Preventive Veterinary Medicine in Hubei Province, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China
    • 3. College of Science, Huazhong Agricultural University, Wuhan 430070, China
    • 4. Hubei Center for Animal Disease Control and Prevention, Wuhan 430070, China

    Abstract: 

    African swine fever (ASF) is an infectious disease caused by African swine fever virus (ASFV) with clinical symptoms of high fever, hemorrhages and high mortality rate, posing a threat to the global swine industry and food security. Quarantine and control of ASFV is crucial for preventing swine industry from ASFV infection. In this study, a recombinase polymerase amplification (RPA)-CRISPR-based nucleic acid detection method was developed for diagnosing ASF. As a highly sensitive method, RPA-CRISPR can detect even a single copy of ASFV plasmid and genomic DNA by determining fluorescence signal induced by collateral cleavage of CRISPR-lwCas13a (previously known as C2c2) through quantitative real-time PCR (qPCR) and has the same or even higher sensitivity than the traditional qPCR method. A lateral flow strip was developed and used in combination with RPA-CRISPR for ASFV detection with the same level of sensitivity of TaqMan qPCR. Likewise, RPA-CRISPR is capable of distinguishing ASFV genomic DNA from viral DNA/RNA of other porcine viruses without any cross-reactivity. This diagnostic method is also available for diagnosing ASFV clinical DNA samples with coincidence rate of 100% for both ASFV positive and negative samples. RPA-CRISPR has great potential for clinical quarantine of ASFV in swine industry and food security.